T. denticola is a frequent component of the complex polymicrobial biofilms that constitute subgingival plaque. It is a "late colonizer" and is proposed to play an etiologic role in the development of periodontal disease. T. denticola ATCC 35405 contains a complete diversity-generating retroelement (DGR) within its 2.8 Mb circular genome. DGRs are a newly discovered family of retroelements that generate diversity in target genes. They function through a template-dependent, reverse trancriptase-mediated mechanism that introduces nucleotide substitutions at specified sites in protein-coding sequences. Variable residues are displayed in the ligand- binding pocket of a specialized C-type lectin scaffold which balances protein diversity with structural stability, creating vast repertoires of receptors for potential ligand-receptor interactions. In addition to a DGR-linked target, the T. denticola genome includes six additional target genes, all of which are predicted to encode surface-exposed lipoproteins. We estimate potential repertoires of 1024 unique polypeptide sequences for each of seven target proteins, far exceeding the estimated capabilities of any naturally occurring protein diversification system described to date. The goal of this R21 is to evaluate the possibility that T. denticola has evolved a DGR-based system to display massively variable protein repertoires on its cell surface. Our specific aims are to: 1) test the hypothesis that the T. denticola DGR is capable of directing site-specific mutagenesis of predicted target genes, and 2) test the hypothesis that diversified target genes encode surface-exposed lipoproteins. DGRs represent a newly discovered mechanism for generating protein diversity and they have the potential to confer powerful selective advantages to their hosts. For T. denticola, we imagine that the accelerated evolution of novel binding interactions could increase the organisms fitness in the highly dynamic microbial community in which it resides. Understanding the DGR system in this species will not only reveal new mechanisms for bacterial evolution, but also provide new paradigms for understanding adaptations that facilitate participation in microbial consortia that play roles in health and disease. PUBLIC HEALTH RELEVANCE: Treponema denticola is a common component of subgingival plaque and is proposed to contribute to the pathogenesis of periodontal disease. Our studies will contribute to an understanding of colonization and virulence mechanisms by this oral bacterium.